Lipid nanoparticle (LNP)-based intravitreal delivery

Abstract

Provided herein are compositions, methods, and systems for the delivery of a CRISPR / Cas-mediated gene editing system to trabecular meshwork cells using lipid nanoparticles (LNPs). Also provided herein are methods, compositions, and systems for treating glaucoma involving reducing the expression of the myocilin (MYOC) gene in trabecular meshwork cells of a patient's eye.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of 35 U.S.C.§ 119(e) of U.S. Provisional Patent Application No. 63 / 353,374, filed on June 17, 2022, and U.S. Provisional Patent Application No. 63 / 417,233, filed on October 18, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Sequence Listing This application is filed with a sequence listing in electronic format. The sequence listing is submitted as a file entitled 80EM - 341773 - WO_SeqList, created on June 16, 2023, and having a size of 479 kilobytes. The information in the electronic format of the sequence listing is hereby incorporated by reference in its entirety.

[0003] The present disclosure generally relates to the fields of molecular biology and biotechnology including gene editing.

Background Art

[0004] Treatment of eye diseases (e.g., glaucoma) requires accurate and efficient intraocular delivery of therapeutic agents, e.g., delivery to the trabecular meshwork cells of a patient. Mutations in the myocilin (MYOC) gene that encodes myocilin are the cause of some forms of juvenile and adult - onset primary open - angle glaucoma (POAG). Myocilin is a 55 - 57 kDa secreted glycoprotein that forms dimers and multimers. It has a myosin - like domain, a leucine zipper region, and an olfactomedin domain. Most of the mutations identified in patients with POAG are located in the olfactomedin domain and are highly conserved across species. In the eye, myocilin is expressed abundantly in the trabecular meshwork (TM), sclera, ciliary body, and iris, and in relatively low amounts in the retina and optic nerve head (Tamm, Prog Retin Eye Res. 2002 Jul; 21(4): 395 - 428).

[0005] Glaucoma is a group of progressive optic neuropathies characterized by the degeneration of retinal ganglion cells, which results in changes at the optic nerve head. There are several types of glaucoma, including POAG, angle-closure glaucoma, congenital glaucoma, and normal-tension glaucoma. The loss of ganglion cells is related to the level of intraocular pressure (IOP), although other factors may also play a role. Reduction of IOP is the only proven method for treating the disease. Initial treatment usually starts with hypotensive eye drops, although laser trabeculoplasty and surgery can also be used to slow the progression of the disease (Weinreb, et al., JAMA. 2014 May 14; 311(18): 1901-1911).

Summary of the Invention

[0006] Disclosed herein is a method for delivering a CRISPR / Cas-mediated gene editing system to cells of a subject's eye, comprising administering to the subject (a) a guide RNA for a target gene or a nucleic acid encoding the guide RNA; and / or (b) a plurality of lipid nanoparticles (LNPs) complexed with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, thereby reducing the expression of the target gene in cells of the subject's eye. For example, the CRISPR / Cas-mediated gene editing system can be delivered to trabecular meshwork cells of the subject. The target gene can be, for example, the myocilin (MYOC) gene (e.g., wild-type MYOC or mutant MYOC gene). In some embodiments, the expression of the target gene, the expression of the protein encoded by the target gene, or both in the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration. In some embodiments, the expression of the target gene is reduced in trabecular meshwork cells of the subject's eye.

[0007] Similarly, disclosed herein are methods for treating a subject having glaucoma, the method comprising administering to the subject (a) a guide RNA targeting the MYOC gene or a nucleic acid encoding the guide RNA; and (b) a plurality of lipid nanoparticles (LNPs) complexed with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, thereby reducing the expression of the MYOC gene in the eye of the subject. In some embodiments, the glaucoma is myocilin-related glaucoma. The glaucoma can be, for example, primary open-angle glaucoma (POAG). In some embodiments, the expression of the MYOC gene is reduced in trabecular meshwork cells of the eye of the subject.

[0008] In the methods described herein, the RNA-guided nuclease can be a Cas9 nuclease, such as Staphylococcus aureus Cas9 (SaCas9) nuclease or Streptococcus pyogenes Cas9 (SpCas9) nuclease. In some embodiments, the site targeted by the guide RNA is within exon 1, exon 2, or exon 3 of the MYOC gene. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. In some embodiments, the guide RNA comprises a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115. The guide RNA can be, for example, a SaCas9 sgRNA or a SpCas9 sgRNA.

[0009] In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 195 to 371. In some embodiments, the guide RNA comprises a nucleotide sequence selected from SEQ ID NOs: 258, 267 to 270, 309, 319, 328 to 331, 370 and 371.

[0010] In some embodiments, one of the plurality of LNPs comprises an ionizable cationic lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG lipid). The LNP may comprise from about 20 to 60% ionizable cationic lipid, from about 18.5% to 60% sterol, from about 0.01 to 30% helper lipid, and / or from about 0% to 10% PEG lipid. In some embodiments, the ionizable cationic lipid is selected from C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA, and DLin-KC2_DMA. In some embodiments, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diundecanoyl phosphatidylcholine (DUPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE, 18:0-18:1 PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoyl phosphatidylglycerol (DOPG), and dipalmitoyl-sn-glycero-3-PG (DPPG).In some embodiments, the sterol is selected from cholesterol, sitosterol, β-sitosterol, phytosterol, fucosterol, zoosterol, and ergosterol. In some embodiments, the sterol is selected from cholesterol, sitosterol, campesterol, stigmasterol, fucosterol, and ergosterol. In some embodiments, the PEG lipid is DMG-PEG, DSG-PEG, PEG ceramide, or PEG phospholipid. In some embodiments, one of the plurality of LNPs comprises about 50 mol% of C12-200, DLIN-MC3, DODMA, or DOTAP, about 10 mol% of DSPC, about 37.0-39.5 mol% of cholesterol or sitosterol, and about 0.5-3.0% of DMG-PEG. In some embodiments, the LNP comprises about 50 mol% of C12-200, about 10 mol% of DSPC, about 37.0-39.5 mol% of sitosterol, and about 0.5-1.5% of DMG-PEG. In some embodiments, the average particle size of the plurality of LNPs is about 80-100 nm, suitably 85-95 nm.

[0011] The plurality of LNPs can be administered to a subject, for example, by intravitreal injection or intracameral injection. In some embodiments, the method comprises a single administration of the plurality of LNPs to the subject. In some embodiments, MYOC expression in the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration. In some embodiments, myocilin protein in the trabecular meshwork cells of the subject's eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after administration.

[0012] As described herein, the subject can be human. In some embodiments, the LNP is complexed separately with (a) a guide RNA or a nucleic acid encoding a guide RNA, and (b) an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease. In some embodiments, the LNP complexed with (a) a guide RNA or a nucleic acid encoding a guide RNA and the LNP complexed with (b) an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease are different LNPs.

[0013] Disclosed herein are guide RNAs targeting the MYOC gene, which include nucleotide sequences specific to fragments in exon 1, exon 2, or exon 3 of the MYOC gene, wherein the guide RNA has a spacer sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 1-27 and 55-115, or a spacer sequence having 1, 2, or 3 mismatches compared to an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 1-27 and 55-115. In some embodiments, the guide RNA includes a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 10. In some embodiments, the guide RNA includes a spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115. In some embodiments, the guide RNA includes a nucleotide sequence selected from SEQ ID NOs: 195-371. In some embodiments, the guide RNA includes a nucleotide sequence selected from SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371.

[0014] Similarly, disclosed herein is a system for treating a subject having glaucoma, the system comprising: (i) gene editing means targeted to reduce the expression of the myocilin (MYOC) gene in the subject's eye; and (ii) lipid nanoparticles (LNPs), wherein the LNPs deliver the gene editing means to the subject's eye. In some embodiments, the glaucoma is myocilin-related glaucoma. The glaucoma can be primary open-angle glaucoma (POAG). In some embodiments, the gene editing means is CRISPR / Cas-mediated gene editing. In some embodiments, the CRISPR / Cas-mediated gene editing comprises a RNA-guided nuclease and a guide RNA targeting a site in the MYOC gene. In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, such as Staphylococcus aureus Cas9 (SaCas9) nuclease or Streptococcus pyogenes Cas9 (SpCas9) nuclease.

[0015] In some embodiments, the site targeted by the guide RNA is within exon 1, exon 2, and exon 3 of the MYOC gene. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some embodiments, the site targeted by the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64, 73, 74, 75, 76, and 115.

[0016] The guide RNA can be a SaCas9 sgRNA or a SpCas9 sgRNA. In some embodiments, the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195 to 371. In some embodiments, the guide RNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267 to 270, 309, 319, 328 to 331, 370 and 371. In some embodiments, the LNP comprises an ionizable cationic lipid, a helper lipid, a sterol, and a poly(ethylene glycol)-lipid (PEG lipid). In some embodiments, the LNP comprises from about 20 to 60% ionizable cationic lipid, from about 18.5% to 60% sterol, from about 0.01 to 30% helper lipid and / or from about 0% to 10% PEG lipid. In some embodiments, the ionizable cationic lipid is selected from C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA, and DLin-KC2_DMA.In some embodiments, the helper lipids are selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diundecanoyl phosphatidylcholine (DUPC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-Sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE, 18:0-18:1 PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoyl phosphatidylglycerol (DOPG) and dipalmitoyl-sn-glycero-3-PG (DPPG).

[0017] In some embodiments, the sterol is selected from cholesterol, sitosterol, phytosterol, fucosterol, animal sterol, and ergosterol. In some embodiments, the sterol is selected from cholesterol, sitosterol, β-sitosterol, campesterol, stigmasterol, fucosterol, and ergosterol. In some embodiments, the PEG lipid is DMG-PEG, DSG-PEG, PEG ceramide, or PEG phospholipid. In some embodiments, the LNP comprises about 50 mol% of C12-200, DLIN-MC3, DODMA, or DOTAP, about 10 mol% of DSPC, about 37.0-39.5 mol% of cholesterol or sitosterol, and about 0.5-3.0% of DMG-PEG. In some embodiments, the LNP comprises about 50 mol% of C12-200, about 10 mol% of DSPC, about 37.0-39.5 mol% of sitosterol, and about 0.5-1.5% of DMG-PEG. In some embodiments, the system is administered to a subject by intravitreal injection or intracameral injection.

[0018] Details of one or more embodiments of the present invention are described below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of some embodiments, and from the appended claims.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0020] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically represent like components unless the context indicates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments are available and other changes can be made without departing from the spirit and scope of the subject matter presented herein. The aspects of the present disclosure generally described herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are clearly contemplated herein and it is readily understood that they form a part of the disclosure herein.

[0021] All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are hereby incorporated by reference in their entirety with respect to the related art.

[0022] Disclosed herein are methods, systems, compositions, and kits for treating a subject having glaucoma. The methods involve reducing the expression of the myocilin gene in trabecular meshwork cells of the subject's eye.

[0023] Definitions As used herein, the term "about" means plus or minus 5% of the indicated value.

[0024] As used herein, the term "RNA-guided endonuclease" refers to a polypeptide that can bind to an RNA (e.g., gRNA) to form a complex that is targeted to a specific DNA sequence (e.g., in a target DNA). Non-limiting examples of RNA-guided endonucleases are Cas polypeptides (e.g., Cas endonucleases, e.g., Cas9 endonuclease). In some embodiments, the RNA-guided endonucleases described herein are targeted to a specific DNA sequence in a target DNA by an RNA molecule to which it binds. The RNA molecule can include sequences that are complementary to and can hybridize to a target sequence within the target DNA, thereby enabling targeting of the binding polypeptide to a specific location within the target DNA.

[0025] As used herein, the term "guide RNA" or "gRNA" refers to a site-specific targeting RNA that can bind to an RNA-guided endonuclease to form a complex and direct the activity of the bound RNA-guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid. A guide RNA can comprise one or more RNA molecules.

[0026] As used herein, the "secondary structure" of a nucleic acid molecule (e.g., an RNA fragment or gRNA) refers to base-pairing interactions within the nucleic acid molecule.

[0027] As used herein, the term "Cas endonuclease" or "Cas nuclease" refers to an RNA-guided DNA endonuclease associated with the CRISPR adaptive immune system.

[0028] Unless otherwise indicated, "nuclease" and "endonuclease" are used interchangeably herein to refer to an enzyme having catalytic activity for nucleotide strand cleavage for polynucleotide cleavage.

[0029] As used herein, the term "constant region" of a gRNA refers to the nucleotide sequence of the gRNA that associates with an RNA-guided endonuclease. In some embodiments, the gRNA comprises a crRNA and a trans-activating crRNA (tracrRNA), where the crRNA and tracrRNA hybridize to each other to form a duplex. In some embodiments, the crRNA comprises, from 5' to 3': a spacer sequence and a minimal CRISPR repeat sequence (also referred to herein as the "crRNA repeat sequence"); the tracrRNA comprises a minimal tracrRNA sequence complementary to the minimal CRISPR repeat sequence (also referred to herein as the "tracrRNA anti-repeat sequence") and a 3' tracrRNA sequence. In some embodiments, the constant region of the gRNA refers to a portion of the crRNA that is the minimal CRISPR repeat sequence and the tracrRNA.

[0030] As used herein, the term "donor template" refers to a nucleic acid strand containing exogenous genetic material that can be introduced into the genome [e.g., by homology-directed repair] to effect targeted integration of the exogenous genetic material. In some embodiments, the donor template may not have regions of homology to the targeted location in the DNA and can be integrated by NHEJ-dependent end joining following cleavage at the target site. The donor template can be DNA or RNA, single-stranded or double-stranded, and can be introduced into cells in linear or circular form.

[0031] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Polynucleotides can be single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids / triple helices or polymers containing purine and pyrimidine bases (e.g., the five biologically occurring bases, adenine, guanine, thymine, cytosine and uracil) or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. In some embodiments, the nucleic acid or polynucleotide refers to any nucleic acid composed of phosphodiester bonds or modified bonds such as phosphotriesters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamidates, carbamates, thioethers, bridged phosphoramidates, bridged methylene phosphonates, bridged phosphoramidates, bridged phosphoramidates, bridged methylene phosphonates, phosphorothioates, methylphosphonates, phosphorodithioates, bridged phosphorothioates or sultone bonds, and combinations of such bonds.

[0032] As used herein, the term "binding" refers to non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be "associated" or "interacting" or "binding" (e.g., when it is stated that molecule X interacts with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner). Binding interactions are characterized by a dissociation constant (Kd), e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15It can be characterized by Kd of M or a number or range between any two of these values, or Kd less than these. Kd may depend on environmental conditions such as pH and temperature. "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a decrease in Kd.

[0033] As used herein, the term "hybridize" or "hybridizing" refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. The pairing can be achieved by any process by which the nucleic acid sequences are linked through base pairing to a substantially or fully complementary sequence such that the nucleic acid sequences form a hybridization complex. "Hybridize" or "hybridizing" can include denaturing the molecule so as to disrupt intramolecular structures (if any) [e.g., secondary structure(s)] within the molecule. In some embodiments, denaturing the molecule includes heating the solution containing the molecule to a temperature sufficient to disrupt the intramolecular structure of the molecule. In some cases, denaturing the molecule includes adjusting the pH of the solution containing the molecule to a pH sufficient to disrupt the intramolecular structure of the molecule. In the context of hybridization, two nucleic acid sequences or segments of a sequence are "substantially complementary" if at least 80% of their individual bases are complementary to each other. In some embodiments, a splint oligonucleotide sequence is not more than about 50% identical to one of two polynucleotides (e.g., RNA fragments) designed to be complementary. The complementary portions of each sequence may be referred to herein as "segments", and segments are substantially complementary if they have 80% or more identity.

[0034] The terms "complementary" and "complementarity" mean that a nucleic acid can form hydrogen bond(s) with another nucleic acid based on the traditional Watson-Crick base pairing rules, i.e., adenine (A) pairs with thymine (U), and guanine (G) pairs with cytosine (C). Complementarity can be perfect (e.g., fully complementary) or imperfect (e.g., partially complementary). Perfect or complete complementarity indicates that each and every nucleobase of one strand can form hydrogen bonds with the corresponding bases in another anti-parallel nucleic acid sequence according to the Watson-Crick standard base pairing. Partial complementarity indicates that only a portion of the consecutive residues of a nucleic acid sequence can form Watson-Crick base pairing with the same number of consecutive residues in another anti-parallel nucleic acid sequence. In some embodiments, complementarity can be at least 70%, 80%, 90%, 100% or a number or range between any two of these values. In some embodiments, complementarity is perfect, i.e., 100%. For example, a complementary candidate sequence segment is fully complementary to the candidate sequence segment, and its sequence can be inferred from the candidate sequence segment using the Watson-Crick base pairing rules.

[0035] As used herein, the term "vector" refers to a polynucleotide construct, typically a plasmid or virus, used to transfer genetic material into a host cell. A vector can be, for example, a virus, plasmid, cosmid or phage. As used herein, a vector can be composed of either DNA or RNA. In some embodiments, the vector is composed of DNA. An "expression vector" is a vector that can direct the expression of a protein encoded by one or more genes carried by the vector when present in an appropriate environment. The vector can preferably replicate autonomously. Typically, an expression vector contains a transcriptional promoter, a gene and a transcriptional terminator. Gene expression is usually under the regulation of a promoter, and the gene is said to be "operably linked" to the promoter.

[0036] As used herein, the terms "transfection" or "infection" refer to the introduction of nucleic acid into a host cell, for example, by contacting the cell with a recombinant MVA virus or gutless picornavirus particle as described herein.

[0037] As used herein, the term "transgene" refers to any nucleotide or DNA sequence that is incorporated into one or more chromosomes of a target cell by human intervention. In some embodiments, the transgene comprises a polynucleotide encoding a protein of interest. The polynucleotide encoding the protein is generally operably linked to other sequences, such as transcriptional control sequences, that are useful to obtain the desired expression of the gene of interest. In some embodiments, the transgene may additionally include a nucleic acid or other molecule(s) used to mark the chromosome into which it is incorporated.

[0038] As used herein, "treatment" refers to a clinical intervention performed in response to a disease, disorder or physiological condition that is present in or is susceptible to a patient. The purposes of treatment include, but are not limited to, alleviation or prevention of symptoms, delay or halt the progression or worsening of a disease, disorder or condition, and / or amelioration of a disease, disorder or condition. "Treatment" refers to one or both of therapeutic treatment and means for prophylaxis or prevention. Subjects in need of treatment include those already suffering from a disease or disorder or undesirable physiological condition and those in whom a disease or disorder or undesirable physiological condition is to be prevented.

[0039] As used herein, the terms "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" refer to an amount sufficient to produce a beneficial or desired biological and / or clinical result.

[0040] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for the administration of the desired compound(s) to a subject. Pharmaceutically acceptable excipients can include substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.

[0041] As used herein, "subject" refers to an animal whose diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. As used herein, "mammal" refers to an individual belonging to the class Mammalia, including but not limited to humans, domestic and farm animals, zoo animals, sport animals, and pets. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates such as monkeys, chimpanzees, and apes, and in particular, humans. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the mammal is not a human. In some aspects, the subject may have or be suspected of having a cardiovascular disease and / or have one or more symptoms of a cardiovascular disease. In some aspects, the subject is a human diagnosed with having a risk of cardiovascular disease at the time of or after diagnosis. In some cases, the diagnosis of having a risk of cardiovascular disease can be determined based on the presence of one or more mutations in the endogenous apolipoprotein(a) (LPA) gene, or genomic sequences near the LPA gene in the genome that can affect the expression of the apo(a) protein.

[0042] The present disclosure provides effective compositions, methods, systems, and means for the delivery of a CRISPR / Cas-mediated gene editing system to trabecular meshwork cells using lipid nanoparticles (LNPs). As described herein, the compositions, methods, systems, and means can be used to treat eye diseases, for example, to treat myocilin-related glaucoma.

[0043] The trabecular meshwork (TM) cells occupy the proximal part of the normal outflow pathway, the main drainage route for aqueous humor derived from the eye, and are the main cell type that forms. TM cells have pore-like structures through which aqueous humor circulates into the Schlemm's canal. In the eye, the myocilin gene (MYOC) is highly expressed in the TM. Wild-type myocilin is secreted into the extracellular matrix (ECM) of the TM, while mutants form aggregates that cause endoplasmic reticulum (ER) stress and TM cell death. Some of the potential pathogenicity of myocilin includes misfolding / unfolding of myocilin; overexpression of myocilin; co-aggregation of Grp94 that restricts autophagy; disruption of ECM homeostasis caused by mutant myocilin; oxidative stress (OS), ER stress and IL-1 / NF-κB inflammatory stress caused by mutant myocilin; and instability resulting from conformational disorders caused by mutant myocilin.

[0044] Accordingly, provided herein are effective therapeutic approaches for delivering a gene editing system to the TM cells of a subject's eye (e.g., treating a glaucoma patient having a mutation in the MYOC gene), including knocking down / reducing the expression of a target gene or a protein encoded by the target gene (e.g., myocilin expression) in the TM cells. In these approaches, mutant and wild-type myocilin alleles are targeted, and the TM cells have the accumulated mutant myocilin removed, reducing ER stress, increasing aqueous humor (AH) outflow, and decreasing intraocular pressure (IOP). Clinical readout information includes measurement of IOP.

[0045] Methods and systems for treating a subject having glaucoma are provided herein, and in some embodiments, it targets knockdown or knockout of the myocilin (MYOC) gene in the subject's eye, and more particularly, in the trabecular meshwork cells of the subject's eye.

[0046] I. System for delivering a CRISPR / Cas-mediated gene editing system to the trabecular meshwork (TM) In one aspect, provided herein are compositions, methods, and systems for delivering a CRISPR / Cas-mediated gene editing system to target TM cells. The compositions, methods, and systems can be used, for example, to treat a subject having glaucoma. Such compositions, methods, and systems can include (i) gene editing means targeting to reduce the expression of a target gene [e.g., myosin (MYOC) gene, ACTA2 gene] in the subject's eye; and (ii) lipid nanoparticles (LNPs) for delivering the gene editing means to the subject's eye.

[0047] Reduction of the expression of a target gene (e.g., MYOC gene, ACTA2 gene) can be achieved through gene editing (including genome editing), a type of genetic manipulation in which nucleotides / nucleic acids are inserted, deleted, and / or substituted in a DNA sequence such as the genome of a target cell. Targeted gene editing enables insertion, deletion, and / or substitution at a preselected site (e.g., in a target gene or target DNA sequence) in the genome of a target cell. For example, when the sequence of an endogenous gene is edited by deletion, insertion, or substitution of nucleotides / nucleic acids, the endogenous gene containing the affected sequence may be knocked out or knocked down due to the sequence change. Thus, targeted editing can be used to disrupt endogenous gene expression. "Targeted integration" refers to a process involving the insertion of one or more exogenous sequences with or without deletion of the endogenous sequence at the insertion site. Targeted integration may result from targeted gene editing if a donor template containing an exogenous sequence is present.

[0048] (a) Genetically edited gene The systems disclosed herein, when delivered to TM cells of a subject's eye, result in disruption of target genes (e.g., the MYOC gene, the ACTA2 gene). As used herein, "gene disruption" refers to a gene that includes an insertion, deletion, or substitution such that expression of a functional protein from the endogenous gene is reduced or inhibited as compared to the endogenous gene. As used herein, "disrupting a gene" refers to a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in the endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting genes are well known to those of skill in the art and are described herein.

[0049] In some embodiments, cells containing a disrupted gene do not express the protein encoded by the gene at a detectable level (e.g., in an immunoassay using an antibody that binds to the encoded protein, or by flow cytometry) (e.g., on the cell surface). Cells that do not express the protein at a detectable level may be referred to as knockout cells. Cells that express the protein at a reduced level may be referred to as knockdown cells.

[0050] (b) MYOC Gene Editing Myocilin is a 55-57 kDa secreted glycoprotein that forms dimers and multimers. It has a myosin-like domain, a leucine zipper region, and an olfactomedin domain. In the eye, myocilin is expressed in large amounts in the TM, sclera, ciliary body, and iris, and in considerably lower amounts in the retina and optic nerve head (Tamm, Prog Retin Eye Res. 2002 Jul; 21(4): 395-428).

[0051] In the present disclosure, disruption of the MYOC gene means that the expression of MYOC in TM cells is substantially reduced / knocked down or completely eliminated. Disruption of the MYOC gene can include editing of one or more genes at one or more suitable target sites (e.g., in the coding region or in non-coding regulatory regions such as the promoter region) that disrupt the expression of the MYOC gene. Such target sites can be identified based on gene editing approaches. Exemplary target sites for gene editing can include exon 1, exon 2, or exon 3 of the MYOC gene or combinations thereof. In some embodiments, editing of one or more genes can occur in exon 1.

[0052] Gene editing can be introduced by gene editing techniques (e.g., CRISPR / Cas technology) using an RNA-guided nuclease and a guide RNA that targets a site in the MYOC gene. In some embodiments, the RNA-guided nuclease can be a Cas9 nuclease including, but not limited to, Staphylococcus aureus Cas9 (SaCas9) nuclease or Streptococcus pyogenes Cas9 (SpCas9) nuclease.

[0053] In some embodiments, the MYOC site targeted by the guide RNA includes any one of the nucleotide sequences listed in Table 1 or Table 2 (see the following Sequence Listing). In some embodiments, the site targeted by the guide RNA includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115. In some specific examples, the site targeted by the guide RNA includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 64, 73, 74, 75, 76, and 115.

[0054] Exemplary MYOC-targeted guide RNAs can be deduced from the target sequences listed in Table 1 or Table 2 and are also within the scope of the present disclosure. In some embodiments, the guide RNA can be a SaCas9 sgRNA or a SpCas9 sgRNA.

[0055] II. Gene editing means Reduction of the expression of target genes (e.g., MYOC gene, ACTA2 gene) in TM cells can be achieved by conventional gene editing methods or the methods described herein.

[0056] (a) Gene editing method Targeted editing can be achieved either through a nuclease-independent approach or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking the exogenous polynucleotide that is introduced into the endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce nucleotide deletions, insertions, or replacements into the endogenous sequence.

[0057] Alternatively, the nuclease-dependent approach can achieve targeted editing at high frequency through the specific introduction of double-strand breaks (DSBs) by specific rare-cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing also utilizes DNA repair mechanisms, such as non-homologous end joining (NHEJ) that occurs in response to DSBs. DNA repair by NHEJ often results in the random insertion or deletion (indel) of a small number of endogenous nucleotides. In contrast to NHEJ-mediated repair, repair can also occur by homology-directed repair (HDR). When a donor template containing exogenous genetic material is present adjacent to a pair of homologous arms, the exogenous genetic material is introduced into the genome by HDR, resulting in targeted integration of the exogenous genetic material.

[0058] In some embodiments, gene disruption results from deletion of genomic sequences using two guide RNAs. Methods using CRISPR-Cas gene editing technology to create genomic deletions in cells (e.g., to knockout genes in cells) are well known (Bauer DE et al. Vis. Exp. 2015; 95:e52118).

[0059] Available endonucleases that can introduce specific and targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR-Cas9 nucleases (CRISPR / Cas9; clustered regularly interspaced short palindromic repeats 9). Additionally, the DICE (dual integrase cassette exchange) system that utilizes phiC31 and Bxb1 integrases can also be used for targeted integration. Some exemplary approaches are disclosed in detail below.

[0060] (b) CRISPR-Cas9 gene editing system The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform for gene editing. It relies on the DNA nuclease Cas9 and two non-coding RNAs, the CRISPR RNA (crRNA) and the trans-activating RNA (tracrRNA), to target DNA cleavage. CRISPR is an abbreviation for clustered regularly interspaced short palindromic repeats, which is a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) similar to exogenous DNA previously exposed to the cell, for example, by a virus that has infected or attacked the prokaryote. These fragments of DNA are used by prokaryotes to detect and destroy similar exogenous DNA, for example, when reintroduced from a similar virus in a later attack. Transcription of the CRISPR locus results in the formation of an RNA molecule containing spacer sequences that associate with and target Cas (CRISPR-associated) proteins that can recognize and cleave foreign, exogenous DNA. Numerous types and classes of the CRISPR / Cas system have been described (see, for example, Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0061] The crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing in a typically 20-nucleotide (nt) sequence in the target DNA. Alteration of the 5'-20 nt sequence in the crRNA enables targeting of the CRISPR-Cas9 complex to a specific locus. The CRISPR-Cas9 complex binds only to a DNA sequence containing a sequence match to the first 20 nt of the crRNA when followed by a specific short DNA motif called the protospacer adjacent motif (PAM) (having the sequence NGG) in the target sequence. The tracrRNA hybridizes to the 3'-end of the crRNA, forming an RNA duplex structure to which the Cas9 endonuclease binds to form a catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

[0062] When the CRISPR-Cas9 complex binds to DNA at the target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, resulting in a double-strand break (DSB) with both strands of the DNA ending in base pairs (blunt ends). The next important step after binding of the CRISPR-Cas9 complex to DNA at the specific target site and formation of the site-specific DSB is repair of the DSB. Cells use two major DNA repair pathways to repair DSBs: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

[0063] NHEJ is a powerful repair mechanism that is thought to be highly active in most cell types, including non-dividing cells. NHEJ is error-prone and can remove or add from one to hundreds of nucleotides at the site of a DSB, such modifications typically being <20nt but often occurring. The resulting insertions and deletions (indels) can disrupt both the coding and non-coding regions of a gene. Alternatively, HDR uses an endogenous or exogenously supplied stretch of homologous donor DNA to repair DSBs with high fidelity. HDR is only active in dividing cells and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as the repair operation.

[0064] (c) Endonucleases for use in CRISPR In some embodiments, a Cas9 (CRISPR-associated protein 9) endonuclease or a nucleotide sequence encoding a Cas9 endonuclease is used in the CRISPR system. The Cas9 enzyme can be from Staphylococcus aureus (SaCas9) or Streptococcus pyogenes (SpCas9), although other Cas9 homologs can also be used. The SaCas9 and SpCas9 DNA sequences are listed in Table 10 (see the Sequence Listing below). It should be understood that wild-type Cas9 may be used, or a modified version of Cas9 provided herein (e.g., a developed version of Cas9 or a Cas9 ortholog or variant) may be used. In some embodiments, Cas9 can be replaced with another RNA-guided endonuclease, such as Cpf1 (from a class II CRISPR / Cas system).

[0065] In some embodiments, the CRISPR / Cas system comprises components from a type I, II, or III system. The most recent classification scheme for CRISPR / Cas loci defines class 1 and class 2 CRISPR / Cas systems as having types I through V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have a single protein effector. The Cas proteins of types II, V, and VI are single proteins, RNA-guided endonucleases, and are referred to herein as "class 2 Cas nucleases." Examples of class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains an RuvC-like nuclease domain.

[0066] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., the Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a class 2 CRISPR / Cas system (such as a single protein Cas nuclease, Cas9 protein, or Cpf1 protein). The protein Cas9 and Cpf1 families are enzymes having DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by designing appropriate guide RNAs further described herein.

[0067] In some embodiments, the Cas nuclease comprises more than one nuclease domain. For example, the Cas9 nuclease can comprise at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB into the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is mutated so that one of the nuclease domains reduces its nucleic acid cleavage activity or is modified to be completely or partially deleted. In some embodiments, the Cas9 nuclease is modified to not contain a functional RuvC-like nuclease domain. In some embodiments, the Cas9 nuclease is modified to not contain a functional HNH-like nuclease domain. In some embodiments where only one of the nuclease domains is functional, the Cas9 nuclease is a nickase that can introduce a single-strand break (“nick”) into the target sequence. In some embodiments, the conserved amino acids within the Cas9 nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease nickase comprises an amino acid substitution in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain include D10A (based on the Streptococcus pyogenes Cas9 nuclease). In some embodiments, the nickase comprises an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the Streptococcus pyogenes Cas9 nuclease).

[0068] In some embodiments, the Cas nuclease is derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of the Cascade complex of the type I CRISPR / Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type VI CRISPR / Cas system.

[0069] (d) Guide RNA (gRNA) CRISPR technology involves the use of a genome-targeting nucleic acid that can direct an endonuclease to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be RNA. The genome-targeting RNA is referred to herein as "guide RNA" or "gRNA". The guide RNA includes at least one spacer sequence that hybridizes to a target nucleic acid sequence within the target gene for editing and a CRISPR repeat sequence.

[0070] In type II systems, the gRNA also includes a second RNA called the tracrRNA sequence. In type II gRNAs, the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In type V gRNAs, the crRNA forms a duplex. In both systems, the duplex binds to a site-specific polypeptide such that the guide RNA and the site-specific polypeptide form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by its association with the site-specific polypeptide. Thereby the genome-targeting nucleic acid directs the activity of the site-specific polypeptide.

[0071] As will be understood by those skilled in the art, each guide RNA is designed to include a spacer sequence that is complementary to a target sequence in the genome. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

[0072] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-stranded molecular guide RNA. The double-stranded molecular guide RNA comprises two strands of RNA molecules. The first strand comprises, in the 5’ to 3’ direction, any spacer extension sequence, spacer sequence, and minimal CRISPR repeat sequence. The second strand comprises a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), 3’ tracrRNA sequence, and any tracrRNA extension sequence.

[0073] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA. A single-molecule guide RNA in a type II system (referred to as “sgRNA”) comprises, in the 5’ to 3’ direction, any spacer extension sequence, spacer sequence, minimal CRISPR repeat sequence, single-molecule guide linker, minimal tracrRNA sequence, 3’ tracrRNA sequence, and any tracrRNA extension sequence. Any tracrRNA extension may comprise elements that contribute to additional functions of the guide RNA (e.g., stability). The single-molecule guide linker links the minimal CRISPR repeat and the minimal tracrRNA sequence so as to form a hairpin structure. Any tracrRNA extension comprises one or more hairpins. A single-molecule guide RNA in a type V system comprises, in the 5’ to 3’ direction, a minimal CRISPR repeat sequence and a spacer sequence.

[0074] The spacer sequence in the gRNA is a sequence (e.g., a 20-nucleotide sequence) that defines the target sequence of the target gene of interest (e.g., a DNA target sequence, a genomic target sequence, etc.). In some embodiments, the spacer sequence ranges from 15 to 30 nucleotides. For example, the spacer sequence may contain 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence contains 20 nucleotides.

[0075] The "target sequence" is in the target gene that is adjacent to the PAM sequence and is the sequence modified by an RNA-guided nuclease (e.g., Cas9). The "target sequence" is in the so-called PAM strand in the "target nucleic acid", which is a double-stranded molecule containing the PAM strand and the complementary non-PAM strand. Those skilled in the art will recognize that the gRNA spacer sequence hybridizes to the complementary sequence located on the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence. The spacer of the gRNA interacts with the target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). Thereby, the nucleotide sequence of the spacer varies depending on the target sequence of the target nucleic acid of interest.

[0076] In the CRISPR / Cas system herein, the spacer sequence is designed to hybridize to the region of the target nucleic acid located 5' of the PAM that is recognizable by the Cas9 enzyme used in the system. The spacer may match the target sequence exactly or may have mismatches. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, Streptococcus pyogenes recognizes a PAM containing the sequence 5'-NRG-3' in the target nucleic acid, where R contains either A or G, N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0077] In some embodiments, the target nucleic acid sequence is 20 nucleotides in length. In some embodiments, the target nucleic acid is less than 20 nucleotides in length. In some embodiments, the target nucleic acid is greater than 20 nucleotides in length. In some embodiments, the target nucleic acid is at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid is at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5' of the first nucleotide of the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNN NGG -3', the target nucleic acid may be the sequence corresponding to the plurality of Ns, where N may be any nucleotide and the underlined NGG sequence is the Streptococcus pyogenes PAM.

[0078] The guide RNAs disclosed herein can target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene are 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene may contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 mismatch.

[0079] For any gRNA sequence provided herein, those that do not explicitly indicate a modification are meant to encompass both the unmodified sequence and sequences having any suitable modification.

[0080] The length of the spacer sequence in any of the gRNAs disclosed herein may similarly depend on the CRISPR / Cas9 system and components used for editing any of the target genes disclosed herein. For example, different Cas9 proteins from different bacterial species have different optimal spacer sequence lengths. Thus, the spacer sequence may have nucleotides of length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or longer than 50. In some embodiments, the spacer sequence may have a length of 18-24 nucleotides. In some embodiments, the targeting sequence may have a length of 19-21 nucleotides. In some embodiments, the spacer sequence may include 20 nucleotides in length.

[0081] In some embodiments, the gRNA can be an sgRNA that may include a 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a variable-length spacer sequence containing 17-30 nucleotides at the 5' end of the sgRNA sequence.

[0082] In some embodiments, the sgRNA does not include uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA includes one or more uracils at the 3' end of the sgRNA sequence. For example, the sgRNA may include 1-8 uracil residues at the 3' end of the sgRNA sequence, such as 1, 2, 3, 4, 5, 6, 7 or 8 uracil residues at the 3' end of the sgRNA sequence.

[0083] Any of the gRNAs disclosed herein that include any of the sgRNAs may be unmodified. Alternatively, it may contain one or more modified nucleotides and / or a modified backbone. For example, a modified gRNA such as an sgRNA may include one or more 2'-O-methyl phosphorothioate nucleotides that may be located at either or both of the 5' end and the 3' end.

[0084] In some embodiments, more than one guide RNA can be used with a CRISPR / Cas nuclease system. Each guide RNA can include a different targeting sequence such that the CRISPR / Cas system cleaves more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties such as activity or stability within the Cas9 RNP complex. If more than one guide RNA is used, each guide RNA can be encoded by the same or different vectors. The promoters used to drive the expression of more than one guide RNA can be the same or different.

[0085] In some embodiments, the gRNAs disclosed herein target the MYOC gene, for example, sites within any one of exons 1-3 of MYOC, for example, exon 1, exon 2, or exon 3 of the MYOC gene. Such a gRNA can include a spacer sequence that is complementary (fully or partially) to a target sequence or a fragment thereof within exon 1 of the MYOC gene. Exemplary target sequences for MYOC are provided in Tables 1-2 (see the Sequence Listing below). Exemplary gRNA sequences can be deduced from the target sequences. In some embodiments, the gRNA targets the ACTA2 gene.

[0086] In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115 or a variant thereof. In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115. In some embodiments, the gRNA comprises a spacer sequence having one, two, or three mismatches to an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115. In some embodiments, the gRNA comprises a spacer sequence having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology to an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115.

[0087] The gRNA spacer sequence is the RNA equivalent of the target sequence. Thus, the gRNA sequence can include a spacer sequence corresponding to any one of SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115 in which "T" is replaced by "U". In some embodiments, the spacer sequence included in the gRNA sequence can be a variant of a spacer sequence having about, at least, or at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% homology to any spacer corresponding to any one of SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115 in which "T" is replaced by "U". In some embodiments, the spacer sequence included in the gRNA sequence can be a variant of a spacer sequence having one, two, or three mismatches compared to any spacer corresponding to any one of SEQ ID NOs: 1-27 and SEQ ID NOs: 55-115 in which "T" is replaced by "U".

[0088] In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 6, 10, 15, 18, and 26, or a variant thereof. In some embodiments, the gRNA comprises a spacer sequence having one, two, or three mismatches to an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 6, 10, 15, 18, and 26.

[0089] In some embodiments, the gRNA comprises a spacer sequence having an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115, or a variant thereof. In some embodiments, the gRNA comprises a spacer sequence having one, two, or three mismatches to an RNA sequence corresponding to any one of the target sequences set forth in SEQ ID NOs: 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109, and 113-115. As an example, guide RNAs or other even smaller RNAs used in the CRISPR / Cas / Cpf1 system are exemplified below and can be readily synthesized by chemical means as described in the art. While chemical synthesis procedures are continuously evolving, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, avoiding the use of gels such as PAGE) tends to become more difficult as the length of the polynucleotide significantly exceeds approximately 100 nucleotides. One approach used to generate longer RNAs is to produce two or more molecules that are ligated together. Longer RNAs, such as those encoding Cas9 or Cpf1 endonucleases, are even more readily generated enzymatically. Various types of RNA modifications, such as those described in the art to enhance stability, reduce the likelihood or extent of the innate immune response, and / or enhance other properties, can be introduced during or after the chemical synthesis and / or enzymatic generation of the RNA.

[0090] In some embodiments, the gRNAs of the present disclosure are produced by in vitro transcription (IVT), synthesis and / or chemical synthesis methods, or combinations thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthesis methods, small region synthesis, and ligation methods are utilized. In some embodiments, the gRNA is made using an IVT enzymatic synthesis method. Methods for making polynucleotides by IVT are well known in the art and are described in WO2013 / 151666. Accordingly, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, used to transcribe the gRNAs described herein in vitro.

[0091] Various types of RNA modifications, such as those described in the art that enhance stability, reduce the likelihood or extent of the innate immune response, and / or enhance other properties, can be introduced during or after the chemical synthesis and / or enzymatic production of the RNA. In some embodiments, non-natural modified nucleobases can be introduced into any of the gRNAs disclosed herein during or after synthesis. In some embodiments, the modifications are in the internucleoside linkages, purine or pyrimidine bases, or sugars. In some embodiments, the modifications are introduced to the ends of the gRNA using chemical synthesis or a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in WO2013 / 052523. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0092] Chemically modified gRNAs can contain one or more phosphorothioated 2'-O-methyl nucleotides at the 3' end and / or 5' end of the gRNA. In some embodiments, the chemically modified gRNA contains a phosphorothioated 2'-O-methyl nucleotide at the 3' end of the gRNA. In some embodiments, the chemically modified gRNA contains a phosphorothioated 2'-O-methyl nucleotide at the 5' end of the gRNA. In some embodiments, the chemically modified gRNA contains 3 or 4 phosphorothioated 2'-O-methyl nucleotides at the 3' end and / or 3 or 4 phosphorothioated 2'-O-methyl nucleotides at the 5' end of the gRNA. In some embodiments, any one of SEQ ID NOs: 18-25 and 26-31 can be chemically modified to have 1, 2, 3, or 4 phosphorothioated 2'-O-methyl nucleotides at the 3' end of the gRNA; 1, 2, or 3 phosphorothioated 2'-O-methyl nucleotides at the 5' end of the gRNA, or a combination thereof.

[0093] The number and position of phosphorothioate bonds can vary. In some embodiments, the bonds can be at positions between the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 6th and 7th, 7th and 8th, 8th and 9th positions from the 5' end of the gRNA; the 9th or 10th or subsequent positions. In some embodiments, the bonds can be at positions between the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 6th and 7th, 7th and 8th, 8th and 9th positions from the 3' end of the gRNA; the 9th or 10th or subsequent positions.

[0094] In some embodiments, the nucleotide analogs / modifications can include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, or xanthosine-5'-triphosphate. Base-modified nucleotides can be 5-methylcytidine-5'-triphosphate,7-Deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-Deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine, 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine, 5’-O-(1-thiophosphate)-guanosine, 5’-O-(1-thiophosphate)-uridine, 5’-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine,It may contain alpha-thio-adenosine, 8-azido-adenosine or 7-deaza-adenosine.

[0095] At least one modified nucleotide and / or at least one nucleotide analogue may include 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, 2'-O-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, inosine, 3-methylcytidine, 2-O-methylcytidine, 2-thiocytidine, N4-acetylcytidine, lysidine, 1-methylguanosine, 7-methylguanosine, 2'-O-methylguanosine, queuosine, epoxyqueuosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, pseudouridine, dihydrouridine, 5-methyluridine, 2'-O-methyluridine, 2-thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine', 5-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, methyl ester of uridine 5-oxyacetic acid, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine or 5-(isopentenylaminomethyl)-2'-O-methyluridine.

[0096] In some embodiments, the chemical modification includes pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine. In some embodiments, the modification includes 2'-O-methyluridine (2'OMe-rU), 2-O-methylcytidine (2'OMe-rC), 2'-O-methyladenosine (2'OMe-rA) or 2'-O-methylguanosine (2'OMe-rG).

[0097] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof to various functional moieties, such as targeting or delivery agents, fluorescent labels, fluids, nanoparticles, etc. The conjugation of polynucleotides and modified polynucleotides is outlined in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0098] In some embodiments, a CRISPR / Cas nuclease system for use in genetically editing any of the target genes disclosed herein comprises at least one guide RNA. In some embodiments, the CRISPR / Cas nuclease system contains multiple gRNAs, e.g., 2, 3, or 4 gRNAs. Such multiple gRNAs can target different sites in the same target gene. Alternatively, multiple gRNAs can target different genes. In some embodiments, the guide RNA(s) and the Cas protein can form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex. The guide RNA(s) can guide the Cas protein to a target sequence(s) on one or more of the target genes disclosed herein, where the Cas protein cleaves the target gene at the target site. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex.

[0099] In some embodiments, the indel frequency (editing frequency) of a particular CRISPR / Cas nuclease system comprising one or more specific gRNAs can be determined using TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing a target gene. In some embodiments, a highly efficient gRNA results in a gene editing frequency higher than 80%. For example, a gRNA is considered highly efficient if it results in a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0100] (e) Other gene editing methods In addition to the CRISPR methods disclosed herein, additional gene editing methods well known in the art may also be used. Some examples include gene editing approaches such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, meganucleases, homing endonucleases, and the like.

[0101] A ZFN is a targeted nuclease that includes a nuclease fused to a zinc finger DNA binding domain (ZFBD), which is a polypeptide domain that binds to DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. Engineered zinc finger domains are non-naturally occurring domains that arise primarily from the application of computer-processed algorithms to rational criteria such as substitution rules and processing information in databases of existing ZFP designs and binding data. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536 and WO03 / 016496. Selected zinc finger domains are non-naturally occurring domains that arise primarily from empirical processes such as phage display, interaction trap, or hybrid selection. ZFNs are described in detail in U.S. Patent Nos. 7,888,121 and 7,972,854. The most recognized example of a ZFN is a fusion of the FokI nuclease and the zinc finger DNA binding domain.

[0102] TALEN is a targeted nuclease that includes a nuclease fused to a TAL effector DNA binding domain. The "transcription activator-like effector DNA binding domain", "TAL effector DNA binding domain" or "TALE DNA binding domain" is the polypeptide domain of the TAL effector protein that causes the binding of the TAL effector protein to DNA. TAL effector proteins are secreted during infection by plant pathogens of the genus Xanthomonas. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA binding domains, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34-amino acid repeats that contain polymorphisms at select repeat positions called repeat variable diresidues (RVDs). TALENs are described in more detail in U.S. Patent Application No. 2011 / 0145940. The most recognized example of a TALEN in the art is a polypeptide in which the FokI nuclease is fused to the TAL effector DNA binding domain.

[0103] Additional examples of targeted nucleases suitable for use as provided herein include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wb / SPBc / TP901-1, whether used individually or in combination.

[0104] Any of the nucleases disclosed herein can be delivered using a vector system including, but not limited to, plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenoviral vectors, poxviral vectors; herpesviral vectors and adeno-associated viral vectors, and combinations thereof.

[0105] Conventional virus- and non-virus-based gene transfer methods can be used to introduce nucleic acids encoding nucleases and donor templates into cells. Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids complexed with delivery media such as liposomes or poloxamers. Viral vector delivery systems include DNA and RNA viruses and can become either episomal or integrated genomes after delivery to cells.

[0106] Methods for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistic particle delivery, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, naked RNA, capped RNA, artificial virions, and enhanced uptake of DNA by drugs. For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for nucleic acid delivery. Some specific examples are provided below.

[0107] III. Delivery of guide RNA and nuclease to TM cells The CRISPR / Cas nuclease system disclosed herein, which includes a guide RNA (gRNA) or a nucleic acid sequence encoding a gRNA and a nucleic acid sequence encoding an RNA-guided nuclease or an RNA-guided endonuclease, can be delivered to TM cells via conventional methods for gene editing of a target gene (e.g., the MYOC gene or the ACTA2 gene). In some embodiments, the components of the CRISPR / Cas nuclease system disclosed herein can be delivered to target cells either separately, simultaneously, or sequentially. In other embodiments, the components of the CRISPR / Cas nuclease system can be delivered together, e.g., as a complex, to the target. In some cases, the gRNA and the RNA-guided nuclease can be pre-complexed together to form a ribonucleoprotein (RNP) that can be delivered to target cells.

[0108] RNP is useful for gene editing because it at least minimizes the risk of random interactions in a nucleic acid-rich cellular environment and protects RNA from degradation. Methods for forming RNP are well known in the art. In some embodiments, an RNP containing an RNA-guided nuclease (e.g., a Cas nuclease, a Cas9 nuclease, etc.) and a guide RNA targeting the MYOC gene can be delivered to TM cells. In some embodiments, the RNP can be delivered to TM cells by electroporation.

[0109] In some embodiments, the RNA-guided nuclease can be delivered to cells with a DNA vector that expresses the RNA-guided nuclease in the cells. In other examples, the RNA-guided nuclease can be delivered to cells in an RNA that encodes the RNA-guided nuclease and expresses the nuclease in the cells. Alternatively or additionally, the gRNA targeting the gene can be delivered to cells as an RNA, or a DNA vector that expresses the gRNA in the cells.

[0110] Delivery of the RNA-guided nuclease, gRNA, and / or RNP can be by direct injection or through cell transfection using well-known methods such as electroporation or chemical transfection. Other cell transfection methods can also be used.

[0111] In some embodiments, one or more nucleic acid sequences and / or polypeptides can be delivered to cells via a virus-based or non-virus-based delivery system, including, for example, an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a lentivirus vector, a herpesvirus vector, nanoparticles, liposomes, lipid nanoparticles, poxviruses, naked DNA administration, plasmids, cosmids, phages, cell encapsulation technology, etc., either in vitro or in vivo.

[0112] In some embodiments, the gRNA and the RNA-guided endonuclease or the nucleic acid encoding the RNA-guided endonuclease, or compositions thereof, can be formulated into liposomes or lipid nanoparticles. In some embodiments, the RNA-guided nuclease and the guide RNA can be delivered to trabecular meshwork cells by lipid nanoparticles (LNPs). The term "lipid nanoparticles" refers to nanoscale particles composed of lipids having a size measured in nanometers (e.g., 1-5,000 nm). The size of the LNPs in the LNP formulations described herein (e.g., CTX-C12-CT LNP formulation) can vary. In some embodiments, the LNP has an average diameter of about, at least, at least about, up to or up to about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm or a number or range between any of these values. In some embodiments, the particle size of the lipid nanoparticles is about 50 to about 200 nm in diameter or about 70 to about 180 nm in diameter or about 80 to about 150 nm in diameter. In some embodiments, the particle size (e.g., average diameter) of the LNP is in the range of 85-95 nm. In some embodiments, the particle size (e.g., average diameter) of the LNP is in the range between about 190 nm, 195 nm, 200 nm, 205 nm or between any two of these values. Without being bound by any particular theory, it is believed that it may be advantageous to use small-sized LNPs to deliver the payload to the trabecular meshwork. For example, to deliver a CRISPR / Cas-mediated gene editing system to target trabecular meshwork cells, it may be advantageous to use LNPs having a size of 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm or a number or range between any two of these values.

[0113] In some embodiments, the lipids included in the lipid nanoparticles comprise a cationic lipid and / or an ionizable lipid. Any suitable cationic lipid and / or ionizable lipid well known in the art can be used to formulate LNPs for the delivery of gRNA and Cas endonucleases into cells. Exemplary cationic lipids include one or more amine group(s) (s) carrying a positive charge. The lipid nanoparticles may further comprise one or more neutral lipids, charged lipids, sterols, tocopherols, hopanoids, and polymer-conjugated lipids, such as poly(ethylene glycol) (PEG)-lipids.

[0114] The LNPs described herein may comprise one or more of the ionizable cationic lipids described herein. For example, the LNP may comprise one or more ionizable cationic lipids selected from the group consisting of C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, or DOTAP. The ionizable cationic lipid may be from about 30 mol% to about 70 mol% (e.g., 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%) of the total lipids present in the LNP. As used herein, "mole percent" refers to the mole percentage of a component compared to the total moles of all lipid components in the LNP (i.e., the total moles of cationic lipids, neutral lipids, sterols, and polymer-conjugated lipids). In some embodiments, the LNP comprises from about 40% to about 60% ionizable cationic lipid of the total lipids in the LNP. For example, the lipid nanoparticles may comprise about 40%, 45%, 50% or 60% ionizable cationic lipid of the total lipids on a molar basis (based on 100% total moles of lipids in the LNP). In some embodiments, the LNP comprises about 50 mole percent of the ionizable cationic lipids described herein.

[0115] The LNPs described herein may further comprise one or more non-cationic lipids (helper lipids). In some embodiments, the LNP may further comprise one or more neutral lipids, charged lipids, sterols, and polymer-conjugated lipids. In some embodiments, the lipid nanoparticles comprise one or more neutral or zwitterionic lipids. The term "neutral lipid" refers to any of several lipid species that exist either uncharged or in a neutral zwitterionic form at physiological pH. The selection of neutral lipids and other non-cationic lipids for use in the particles described herein is generally guided by, for example, consideration of lipid particle size and the stability of the lipid particles in the bloodstream. In some embodiments, the non-cationic lipid contains a saturated fatty acid having a carbon chain length in the range of C 10 to C 20 . In some embodiments, C 10 to C 20A non-cationic lipid containing a monovalent or divalent unsaturated fatty acid having a carbon chain length within the range is used. Additionally, a non-cationic lipid containing a mixture of saturated and unsaturated fatty acid chains may be used. Suitable neutral lipids include, but are not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (DMPC), distearoyl-phosphatidylethanolamine (DSPE), SM, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol or mixtures thereof. In some embodiments, the helper lipid is a PC class lipid [e.g., DLPC(12:0), DMPC(14:0), DPPC(16:0), DSPC(18:0), DOPC(18:1), DUPC(18:2), POPC(16:0, 18:1), SOPC(18:0, 18:1)]; a PE class-like lipid [e.g., DOPE(18:1), DSPE(18:0), DPPE(16:0), DMPE(14:0) SOPE(18:0, 18:1), POPE(16:0, 18:1)]; a PG class-like lipid [e.g., DOPG(18:1), DPPG(16:0)] or a mixture thereof or including them.In some embodiments, the helper lipid is 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), DMPC, DPPC, DSPC, DOPC, diundecanoyl phosphatidylcholine (DUPC), POPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), DOPE, DSPE, DPPE, DMPE, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE, 18:0-18:1 PE), POPE, dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl-sn-glycero-3-PG (DPPG) or a mixture thereof or includes these.

[0116] In some embodiments, the neutral lipid can be from about 5 mol% to about 20 mol% (e.g., about 5 mol%, 10 mol%, 15 mol%, 20 mol%) of the total lipids present in the LNP. In some embodiments, the LNP contains neutral lipids that are about 10% or more of the total lipids in the LNP on a molar basis (based on 100% total moles of the lipids in the LNP).

[0117] The LNP may further contain sterols, such as cholesterol. The sterol may be from about 10 mol% to about 60 mol% of the total lipids present in the LNP, optionally from about 20 mol% to about 50 mol%, and further optionally from about 30% to about 40%. In some embodiments, the sterol is about 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol% or 50 mol% of the total lipids present in the LNP. In the LNP described herein, the sterol can be one or more of cholesterol, sitosterol, campesterol, plant sterols (phytosterols, also referred to as stigmasterol, β-sitosterol, etc.), sterols derived from algae (e.g., fucosterol), sterols derived from animals (also referred to as "animal sterols"), and sterols derived from fungi and protozoa (e.g., ergosterol). The LNP disclosed herein may contain the classes of compounds tocopherols and hopanoids (diproptene and diproptenol). In some embodiments, the classes of compounds tocopherols and hopanoids (diproptene and diproptenol) are for replacing the sterols in the LNP. In some embodiments, the classes of compounds tocopherols and hopanoids (diproptene and diproptenol) are present in the LNP in addition to the sterols.

[0118] The LNP may further comprise a polymer-conjugated lipid, such as a polyethylene glycol (PEG)-modified lipid. Exemplary PEG-conjugated lipids include, for example, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEG-dimyristoyl glycerol (DMG), or mixtures thereof. In some embodiments, the PEG-conjugated lipid can be from about 0 mol% to about 10 mol% of the total lipids in the LNP. For example, the PEG-conjugated lipid can be about 0 mol%, 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol% or 10 mol% of the total lipids present in the LNP. In some embodiments, the polymer-conjugated lipid (e.g., PEG-conjugated lipid) is from about 0 mol% to about 5 mol% (e.g., 0.5%, 1%, 1.5%, 2%, 2.5%, 3%) of the total lipids present in the LNP. The PEG-modified lipid can be, for example, DMG-PEG, DSG-PEG, PEG-ceramide, PEG-phospholipid, or combinations or mixtures thereof.

[0119] In some embodiments of the LNPs described herein, the ionizable cationic lipid can be C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA or DOTAP; the helper lipid can be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); the sterol can be cholesterol or sitosterol; and the PEG lipid can be DMG-PEG.

[0120] In some embodiments, the LNP comprises about 50 mol% C12-200, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA and / or DOTAP, about 10 mol% DSPC, about 37.0 - 39.5 mol% cholesterol or sitosterol and about 0.5 - 3.0% DMG-PEG. In some specific examples, the LNP comprises about 50 mol% C12-200, about 10 mol% DSPC, about 37.0 - 39.5 mol% sitosterol and about 0.5 - 1.5% DMG-PEG.

[0121] The LNP can be administered by any suitable route that results in delivery to the patient's eye. For example, an effective amount of the LNP can be administered to the patient by intravitreal injection and / or intracameral injection.

[0122] IV. Compositions and therapeutic applications The Cas9 gRNA system disclosed herein can be administered to a subject for therapeutic purposes, such as the treatment of myocilin-related glaucoma. Disruption of the MYOC gene knocks down / reduces myocilin expression in trabecular meshwork (TM) cells, which then results in clearance of the accumulated mutant myocilin, thereby reducing ER stress, increasing aqueous humor (AH) outflow, and decreasing intraocular pressure (IOP).

[0123] Accordingly, provided herein is a method for treating a subject having glaucoma, the method comprising reducing the expression of the myocilin (MYOC) gene in TM cells of the subject's eye. In some embodiments, the method for treating a subject having glaucoma comprises: (a) a guide RNA targeting the myocilin (MYOC) gene or a nucleic acid encoding the guide RNA; and (b) administering to the subject a plurality of lipid nanoparticles (LNPs) complexed with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease, thereby reducing the expression of the MYOC gene in the subject's eye. In some embodiments, the glaucoma can be myocilin-related glaucoma. In some embodiments, the glaucoma can be POAG. In some embodiments, the expression of the MYOC gene can be reduced in TM cells of the subject's eye.

[0124] As described above and herein, the MYOC gene can be disrupted in TM cells by a CRISPR / Cas-mediated gene editing system comprising an RNA-guided nuclease (or a nucleotide sequence encoding an RNA-guided nuclease) and a guide RNA targeting a site in the MYOC gene (e.g., exon 1, 2, or 3 of the MYOC gene). In some embodiments, the CRISPR / Cas-mediated gene editing system can be provided in a pharmaceutical composition. Thus, the composition can comprise one or more gRNAs (MYOC gRNAs), an RNA-guided endonuclease described herein, or a nucleotide sequence encoding an RNA-guided endonuclease. The MYOC gRNA can be any gRNA described herein or a variant thereof that targets one or more of the target sequences of SEQ ID NOs: 1-27 and 55-115. The RNA-guided endonuclease or the nucleotide sequence encoding an RNA-guided endonuclease can be any RNA-guided endonuclease described herein. In some embodiments, the DNA endonuclease is Cas9. In some embodiments, the Cas9 endonuclease is Streptococcus pyogenes (SpCas9) nuclease. In some embodiments, the Cas9 endonuclease is Staphylococcus aureus Cas9 (SaCas9) nuclease.

[0125] In some embodiments, the RNA-guided nuclease and the guide RNA are delivered to trabecular meshwork cells by lipid nanoparticles (LNPs). The LNPs can include one or more ionizable cationic lipids selected from C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, or DOTAP; helper lipids of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); sterols of cholesterol and / or sitosterol; and PEG lipids of DMG-PEG. The LNPs can include one or more PEG glycerides, such as PEG glycerides of the DMG-PEG and DSG-PEG classes. In some embodiments, the LNPs include one or more DSG-PEG. The LNPs can include one or more PEG ceramides, such as C16-PEG 2000 ceramide or C8 PEG2000 ceramide; one or more PEG phospholipids, such as 14:0 PEG 2000 PE; or any combination thereof. In some embodiments, the LNPs include PEG ceramide. In some embodiments, the LNPs include PEG phospholipid.

[0126] In some embodiments, the LNPs include about 50 mol% of C12-200, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, and / or DOTAP, about 10 mol% of DSPC, about 37.0-39.5 mol% of cholesterol or sitosterol, and about 0.5-3.0% of DMG-PEG. In some specific examples, the LNPs include about 50 mol% of C12-200, about 10 mol% of DSPC, about 37.0-39.5 mol% of sitosterol, and about 0.5-1.5% of DMG-PEG.

[0127] In some embodiments, the compounds of the compositions described herein are encapsulated in the lipid portion of the lipid nanoparticles or in the aqueous portion encapsulated by a part or all of the lipid portion of the lipid nanoparticles. Encapsulation may be complete encapsulation, partial encapsulation, or both. In some embodiments, the nucleic acid and / or polypeptide is completely encapsulated in the lipid nanoparticles.

[0128] In some embodiments, one or more of the compounds described herein associate with liposomes or lipid nanoparticles via covalent or non-covalent bonds. In some embodiments, any of the compounds in the composition can be contained in liposomes or lipid nanoparticles separately or in combination.

[0129] The LNP can be administered by any suitable route that results in delivery to the patient's eye. For example, an effective amount of the LNP can be administered to the patient by intravitreal injection and / or intracameral injection.

[0130] Effective amount refers to the amount of LNP required to achieve a level of modulation to prevent or alleviate at least one or more signs or symptoms of a medical condition and relates to a sufficient amount of the composition for treating a subject having the desired effect, e.g., treating a medical condition. Effective amount also includes an amount sufficient to prevent or delay the occurrence of symptoms of the disease, alter the course of symptoms of the disease (e.g., but not limited to, slow the progression of symptoms of the disease), or reverse the symptoms of the disease. It is understood that for any given case, a suitable effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0131] The effectiveness of a treatment using the means disclosed herein can be determined by a skilled clinician. A treatment is considered "effective" if, for example, any one or all of the signs or symptoms of the level of a functional target change in a beneficial manner (e.g., increase by at least 10%), or if other clinically observable symptoms or markers of a disease (e.g., glaucoma) improve or resolve. Effectiveness can also be measured by preventing the worsening of a subject as evaluated by the need for hospitalization or medical intervention (e.g., the progression of the disease stops or at least slows down). Methods for measuring these indicators are well known to those of skill in the art and / or are described herein. A treatment includes any treatment of a disease in a subject: (1) suppressing the disease, e.g., stopping or slowing the progression of symptoms; or (2) reducing the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the occurrence of symptoms.

[0132] In some embodiments, the plurality of LNPs are administered (e.g., locally) to a subject at a dose of about 0.1 to 5 mg / kg [determined by the total nucleic acid (e.g., the total of the target gene gRNA (e.g., MYOC gRNA) and Cas9 mRNA)], which includes 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg or 5 mg / kg or a number or range between any two of these values. In some embodiments, the plurality of LNPs are administered to a subject at a dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg or 3.0 mg / kg or at about these doses [determined by the total of the target gene gRNA (e.g., MYOC gRNA) and Cas9 mRNA in some embodiments]. In some embodiments, the plurality of LNPs are administered at a dose of about 40 μg per eye, 45 μg per eye, 50 μg per eye, 55 μg per eye, 60 μg per eye, 65 μg per eye, 70 μg per eye, 75 μg per eye, 80 μg per eye, 85 μg per eye, 90 μg per eye, 95 μg per eye, 100 μg per eye or a number or range between any two of these values.

[0133] In some embodiments, the compositions described above may further include one or more additional reagents, such reagents being selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, wash buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro production of polypeptides from DNA, adapters for sequencing, etc. The buffer can be a stabilization buffer, a reconstitution buffer, a dilution buffer, etc. In some embodiments, the composition may also include one or more components that can be used to facilitate or enhance on-target binding or DNA cleavage by endonucleases, or to improve the specificity of targeting.

[0134] In some embodiments, any component of the composition is formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the specific mode of administration and dosage form. In embodiments, the guide RNA composition is generally formulated to achieve a range of from about pH 3 to about pH 11, from about pH 3 to about pH 7, depending on the physiologically compatible pH, formulation, and route of administration. In some embodiments, the pH is adjusted to a range of from about pH 5.0 to about pH 8.

[0135] Suitable excipients can include carrier molecules containing large, slowly metabolized macromolecules such as, for example, proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactivated virus particles. Other exemplary excipients include antioxidants (e.g., without limitation, ascorbic acid), chelating agents (e.g., without limitation, EDTA), carbohydrates (e.g., without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (e.g., without limitation, oils, water, saline, glycerin, and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

[0136] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no other materials in addition to the active ingredient and water, or sterile aqueous solutions that contain a buffer such as sodium phosphate at physiological pH values, contain saline, or contain both, such as phosphate buffered saline. Aqueous carriers can contain more than one buffering salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol and other solutes. Also, the liquid composition can contain a liquid phase in addition to water and excluding water. Exemplary such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in a cell composition that is effective in the treatment of a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0137] In some embodiments, the compounds described herein of the composition (e.g., an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and / or gRNA) can be delivered via transfection, e.g., calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, electro-nuclear transport, chemical transduction, electrotransduction, lipofectamine mediated transfection, Effectene mediated transfection, lipid nanoparticle (LNP) mediated transfection or any combination thereof. In some embodiments, the composition is introduced into cells via lipid-mediated transfection using lipid nanoparticles.

[0138] The compositions described herein can be administered to a subject in need of treatment for an eye disease (e.g., glaucoma). Accordingly, the present disclosure also provides a gene therapy approach for treating glaucoma in a patient by reducing the expression of the MYOC gene (e.g., wild-type MYOC gene or mutant MYOC gene) in TM cells of the subject's eye. In some embodiments, a method for treating a subject having a certain type of glaucoma is disclosed. The method includes reducing the expression of the myocilin (MYOC) gene in the subject's eye. In some embodiments, the method includes administering to the subject (a) a guide RNA (gRNA) targeting the MYOC gene or a nucleic acid encoding the gRNA, and (b) a plurality of LNPs complexed with a nucleic acid encoding an RNA-guided endonuclease or an RNA-guided endonuclease, thereby reducing glaucoma.

[0139] A subject can be administered a plurality of nanoparticles once. In some embodiments, a subject can be administered the plurality of nanoparticles two or more times, such as two times, for treatment. The two administrations of nanoparticles to a subject can be separated by a suitable period. In some embodiments, the suitable period is one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, three months, four months, five months, six months, one year, two years, three years or longer, or about these periods. In some embodiments, two of the two or more administrations are separated by about two weeks to about two months, such as about three weeks. In some embodiments, each two of the two or more administrations are separated by about two weeks to about two months, such as about three weeks. In some embodiments, two of the two or more administrations are separated by about one month to about four months, such as about two months or three months or longer. In some embodiments, each two of the two or more administrations are separated by about one month to about four months, such as about two months or three months. In some embodiments, two of the two or more administrations are separated by at least two months or three months. In some embodiments, each two of the two or more administrations are separated by at least two months or three months. In some embodiments, the expression level of a target gene (e.g., the MYOC gene) in a subject (e.g., in the eye of the subject) that has received a single administration of the composition described herein is substantially (e.g., at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) reduced and maintained at the reduced level for at least two months, three months, four months, six months, ten months, one year, eighteen months, two years, three years, four years, five years, ten years, fifteen years, twenty years or longer after administration.In some embodiments, the level of the protein encoded by the target gene (e.g., the myosin protein level) in a subject (e.g., in the subject's eye) that has received a single administration of the composition described herein may be substantially reduced (e.g., by at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and maintained at the reduced level for at least 2 months, 3 months, 4 months, 6 months, 10 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years or longer after administration. The preferred period between two administrations may be the same as or different from the preferred period between another two administrations.

[0140] In some embodiments, the target tissue for the compositions and methods described herein is the trabecular meshwork tissue. In some embodiments, the target cells for the compositions and methods described herein are trabecular meshwork cells.

[0141] In some embodiments, these pharmaceutical compositions can be administered by any suitable route capable of delivering the compound to the target tissue / cell. For example, the pharmaceutical composition can be delivered via intravitreal, intracameral, subconjunctival, sub-Tenon's, retrobulbar, topical, suprachoroidal and / or posterior juxtascleral administration. In some embodiments, the pharmaceutical composition is administered to the subject by intravitreal injection or intracameral injection. The administration may be topical. In some embodiments, more than one administration over periods of various intervals, e.g., daily, weekly, monthly or annually, may be used to achieve the desired level of gene expression.

[0142] Subjects in need of treatment may have myocilin-related glaucoma. Myocilin-related glaucoma includes types of glaucoma associated with changes in the myocilin gene (MYOC). In some embodiments, myocilin-related glaucoma includes open-angle glaucoma (OAG). In some embodiments, the OAG is primary OAG (POAG). In some embodiments, the OAG is juvenile-onset OAG (JOAG).

[0143] In some embodiments, the expression of the target gene (e.g., MYOC) in the eye of the subject after administration is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some embodiments, the expression of the protein encoded by the target protein (e.g., myocilin protein, α-SMA protein) in the trabecular meshwork cells of the eye of the subject after administration is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. The reduction is a comparison with the myocilin expression or the concentration of myocilin protein in the trabecular meshwork cells of the subject (e.g., mammalian, NHP, human subject) before administration of the plurality of nanoparticles. In some embodiments, the reduction is a comparison with the expression of the target gene or the protein encoded by the target gene (e.g., MYOC expression or the concentration of myocilin protein) in one or more untreated subjects. In some embodiments, the reduction is a comparison with a reference level of the expression of the target gene (e.g., MYOC expression) or the concentration of the protein encoded by the target gene (e.g., myocilin protein) in healthy and / or unmodified subjects.

[0144] In some embodiments, the method may further include measuring intraocular pressure in a subject before, during, and / or after administration. In some embodiments, the method includes identifying a subject in need of treatment. In some embodiments, a subject in need of treatment may be identified as having an elevated intraocular pressure (IOP). The compositions and methods described herein can reduce IOP in a subject by at least about 20%, at least about 40%, at least about 70%, or at least about 90% after administration.

[0145] Combination therapies are also encompassed by the present disclosure. For example, the means disclosed herein can be used in combination with other therapeutic agents for treating the same indication, or for enhancing the effectiveness of MYOC gene editing in TM cells, and / or for reducing the side effects of MYOC gene editing in TM cells.

[0146] V. Kit The present disclosure also provides kits for therapeutic use. In some embodiments, the kits provided herein can include components for performing gene editing of the MYOC gene in TM cells. Components for genetically editing the MYOC gene can include a suitable endonuclease, such as an RNA-guided endonuclease, and a nucleic acid guide that directs cleavage of one or more suitable genomic sites by the endonuclease. For example, the kit can include an mRNA encoding a Cas enzyme, such as Cas9, and one or more gRNAs targeting MYOC. Any of the gRNAs specific for the MYOC gene can be included in the kit.

[0147] Any of the kits disclosed herein can further include instructions for use. In some embodiments, the included instructions include instructions for use of the gene editing components for genetically editing the MYOC gene.

[0148] In some embodiments, the kits disclosed herein may include instructions for the administration of the LNPs disclosed herein to achieve the desired therapeutic effect. Alternatively or additionally, the kit may further include instructions for selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment. Instructions for use generally include information on dosage, dosing schedule, and route of administration for the desired treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or less than a unit dose. The instructions provided with the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the contents / components of the kit are used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0149] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packages, etc. Also contemplated is packaging for use in combination with a specific device, e.g., an infusion device for administration of the contents. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be penetrated by a hypodermic needle). The container may also have a sterile access port.

[0150] The kit may optionally provide additional components, such as buffers and explanatory information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the present disclosure provides a manufactured product comprising the contents of the kit described above.

[0151] General Techniques In the practice of the present disclosure, unless otherwise indicated, conventional techniques within the skill of the art are used in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology. Such techniques are described in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.1994);Current Protocols in Immunology (J. E. Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999);The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995);DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985);Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985. 》 ;Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984 》 ;Animal Cell Culture (R.I. Freshney, ed. (1986 》 ;Immobilized Cells and Enzymes (lRL Press, (1986 》; and are well described in B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0152] Even without further details, those skilled in the art are considered to be able to make maximum use of the present invention based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative and in no case are to be construed as limitations of the parts not disclosed in the present disclosure. All publications cited herein are incorporated by reference for the purposes or subject matters referred to herein.

Example

[0153] Example 1: In vitro sgRNA screening This example describes the screening of guide RNAs targeting MYOC by CRISPR / Cas-mediated gene editing.

[0154] HEK293T cells were used for in vitro sgRNA screening and nucleofected with Cas9 (SaCas9 or SpCas9) and gRNA ribonucleoprotein complexes. Genomic DNA was extracted, specific DNA sequences were PCR amplified, and subjected to tracking of indels by decomposition (TIDE) analysis. Both the total and frameshift insertion / deletion (indel)% were reported in Table 6 (for SaCas9) and Table 7 (for SpCas9). The frameshift indel% refers to indels (i.e., ±1nt, ±2nt, ±4nt) predicted to cause frameshift mutations in the MYOC coding sequence. Therefore, the frameshift indel% refers to the subtraction of the frequency of indels such as ±3nt, ±6nt, ±9nt, etc. from the frequency of total indels.

[0155]

Table 1

[0156] The results of quantifying the editing efficiency of SaCas9 sgRNAs targeting the MYOC coding sequence are shown in Figures 1A - 1B. Figure 1A illustrates the quantification ranked by guide number, and Figure 1B illustrates the quantification ranked by the percentage of indels. As shown, a ribonucleoprotein complex of SaCas9 protein and one of 27 SaCas9 sgRNAs was transfected into HEK293T cells, and the cells were collected at a later time point. After DNA extraction, PCR and TIDE were performed to determine the percentage of indels at the cleavage site. The total indel percentage ranged from 89.65% at SaMCh10 to 1.48% at SaMCh4 under the tested conditions. The highest frameshift percentage (80.83%) was also seen in the sample transfected with SaMCh10 sgRNA.

[0157]

Table 2 - 1

Table 2 - 2

[0158] The results of quantifying the editing efficiency of SpCas9 sgRNAs targeting the MYOC coding sequence are shown in Figures 2A - 2B. Figure 2A illustrates the quantification ranked by guide number, and Figure 2B illustrates the quantification ranked by the percentage of indels. As shown, a ribonucleoprotein complex of SpCas9 protein and one of 61 SpCas9 sgRNAs was transfected into HEK293T cells, and the cells were collected at a later time point. After DNA extraction, PCR and TIDE were performed to determine the percentage of indels at the cleavage site. The total editing percentage ranged from 91.60% at SpMCh47 to 1.98% at SpMCh58 under the tested conditions. The highest frameshift percentage was 84.23% (85.24% total indels) and was observed in the sample transfected with SpMCh10 sgRNA.

[0159] Example 2: In vitro lipid nanoparticle screening This example describes an in vitro efficacy study of lipid nanoparticle (LNP) delivery via various LNPs.

[0160] A plurality of human primary cells and one cell line, including primary trabecular meshwork (TM) cells and an immortalized glaucomatous TM cell line (GTM3) expressing myosin mutant (G364V) protein fused with dsRED, were used for this study. All LNPs were formulated with GFP mRNA and delivered to cells. Photographs were taken at the same time point for all formulations tested, and GFP scores were determined using no GFP signal as score 0 and the highest GFP signal as score 3.

[0161] The characteristics and formulations of the LNPs used in these studies are shown in Table 8 (LNPs obtained from Source 1) and Table 9 (LNPs obtained from Source 2).

[0162]

Table 3

[0163]

Table 4

[0164] The results are shown in Figures 2A / B - 6A / B and Figure 7. Specifically, Figures 3A - 3B illustrate the screening of LNPs obtained from Source 1 in primary TM cells. LNPs carrying GFP mRNA were transfected into primary TM cells in culture. Twenty-two hours after transfection, photographs were taken (Figure 3A), and GFP scores were determined (Figure 3B).

[0165] Figures 4A - 4B illustrate the screening of LNPs obtained from Source 2 in the GTM3 cell line. LNPs carrying GFP mRNA were transfected into immortalized glaucomatous TM cells in culture. The GTM3 cells harbored a transgenic form of mutant G364V myosin fused to dsRED (photographs not shown). Twenty - two hours after transfection, photographs were taken (Figure 4A) and GFP scores were determined (Figure 4B).

[0166] Figures 5A - 5B illustrate the screening of LNPs obtained from Source 2 in primary TM cells obtained from Source 1. LNPs carrying GFP mRNA were transfected into primary TM cells in culture. Twenty - two hours after transfection, photographs were taken (Figure 5A) and GFP scores were determined (Figure 5B).

[0167] Figures 6A - 6B illustrate the screening of LNPs obtained from Source 2 in primary TM cells obtained from Source 2. LNPs carrying GFP mRNA were transfected into primary TM cells in culture. Twenty - two hours after transfection, photographs were taken (Figure 6A) and GFP scores were determined (Figure 6B).

[0168] TM cells are notoriously difficult to isolate and other cell types may be contaminated, so several different sources of trabecular meshwork (TM) cells were used for LNP screening. By using multiple sources, patterns could be confirmed regarding LNP transfection efficacy in TM cells.

[0169] Figure 7 illustrates the results of quantification of the editing efficiency of selected SpCas9 sgRNAs targeting the MYOC coding sequence, using lipid nanoparticles (LNPs) selected for delivery in GTM3 cells. GTM3 cells were transfected with either one of two versions of CTX-C12-200-CT from Source 1: (1) containing 1 mol% DMG-PEG; or (2) containing 1.5 mol% DMG-PEG. Both versions were formulated with Cas9 protein and SpMCh21 sgRNA. Two different doses were transfected into GTM3 cells, which were collected 2 days after transfection, DNA was extracted, and PCR and TIDE were performed to determine the total indel and frameshift indel percentages at both the genomic and transgenic sites. At the low dose, 1 mol% DMG-PEG worked slightly better than 1.5 mol% DMG-PEG. However, at the high dose, they worked similarly, suggesting a plateau effect at such doses. The experiment was for confirming the effectiveness of LNP formulations that worked outstandingly well among those tested with Cas9 / sgRNA to achieve editing in an in vitro cell model.

[0170] Example 3: In vivo lipid nanoparticle screening This example describes the screening of LNPs in vivo. BALB / c mice were used for the in vivo study. One microliter containing LNP formulated with GFP mRNA was injected by intravitreal injection, and the whole globe was isolated at a later time point (e.g., 24-hour time point). The whole globe was fixed, embedded, and processed for immunohistochemical staining for GFP expression. Photographs were taken at the same time point for all formulations tested, and the GFP score was determined using no GFP signal as score 0 and the highest GFP signal as score 3.

[0171] Figure 8 illustrates the GFP score of the LNP obtained from Source 1 of the mouse fibrovascular trabecular meshwork after intravitreal injection of 300 ng of LNP. LNP carrying GFP mRNA was injected intravitreally into the eyes of BALB / c mice. At 5 hours, the mice were euthanized and their eyes were processed for IHC and anti-GFP staining. Photographs were taken and the GFP score was determined.

[0172] Figure 9 illustrates the GFP score of the LNP obtained from Source 2 of the mouse fibrovascular trabecular meshwork after intravitreal injection of 300 ng of LNP. LNP carrying GFP mRNA was injected intravitreally into the eyes of BALB / c mice. At 5 hours, the mice were euthanized and their eyes were processed for IHC and anti-GFP staining. Photographs were taken and the GFP score was determined.

[0173] Figure 10A illustrates the IHC GFP staining of the delivery of CTX-C12-CT (described in Table 8) obtained from Source 1 of the mouse fibrovascular trabecular meshwork after intravitreal injection of 300 ng of LNP. GFP expression was seen in the fibrovascular trabecular meshwork tissue of cross-sections of the eyes of Balb / c mice at 5 hours after injection of CTX-C12-CT formulated with GFP mRNA. Figure 10B illustrates the GFP protein expression in the mouse fibrovascular trabecular tissue after intravitreal injection of LNP CTX-C12-200-CT / eGFP mRNA into the mouse eye. Figure 10C illustrates the GFP protein expression in the mouse fibrovascular trabecular tissue after intravitreal injection of LNP A14 / eGFP mRNA into the mouse eye. These results are a demonstration of the concept that intravitreal injection of LNP in the eye results in the expression of cargo in the fibrovascular trabecular meshwork tissue.

[0174] Example 4: In vitro LNP delivery of Cas9 and sgRNA This example describes the in vitro LNP delivery of Cas9 mRNA and sgRNA. Immortalized human glaucomatous TM cell lines expressing myosin mutants (G364V or Y437H) protein as a fusion with dsRED were transfected with LNP formulated with Cas9 mRNA and sgRNA targeting MYOC. A CTX-C12-CT formulation containing either 1% or 1.5% DMG-PEG LNP was used to deliver Cas9 mRNA and sgRNA.

[0175] Primary human trabecular meshwork cells were transfected for 2 days with LNP formulated with Cas9 mRNA and sgRNA targeting MYOC, then the cells were treated with dexamethasone to induce myosin expression and harvested 3 days later.

[0176] Genomic DNA was extracted and subjected to TIDE (Tracking of Indels by DEcomposition) analysis. The results indicate that the combination of one of the lead sgRNAs (SpMCh21) and one of the best LNPs (CTX-C12-CT) can achieve high levels of editing in trabecular meshwork cells.

[0177] Figure 11 demonstrates MYOC gene editing and myosin protein knockdown after delivery of LNP CTX-C12-200-CT / Cas9 mRNA / SpMCh10 sgRNA to human glaucomatous trabecular meshwork cell line GTM3 MYOC Y437H -dsRED. In this study, human glaucomatous GTM3 MYOC Y437H -dsRED was transfected with various amounts of LNP CTX-C12-200-CT formulated with Streptococcus pyogenes Cas9 mRNA and SpMCh10 targeting the human MYOC gene. GTM3 MYOC Y437H -dsRED cell line is an immortalized human trabecular meshwork cell line that stably expresses the transgene myosin mutant Y437H fused to the reporter protein dsRED.

[0178] Targeting the myocilin mutant sequence affects the expression of the fusion protein, as shown by the reduction of dsRED expression in the edited samples observed by epifluorescence microscopy (Figure 11, Panel A). Proteins were extracted 5 days after transfection, and immunoblots were performed using anti-GAPDH and anti-myocilin to detect myocilin fused with dsRED (Figure 11, Panels B and C). DNA was also extracted, and PCR and TIDE were performed to determine the percentage of indels at the cleavage sites for both the genomic and transgenic loci (Figure 11, Panel D). Myocilin mutant-dsRED protein expression was associated with the transgenic expression of MYOC, while the genomic MYOC was also edited and wild-type expression of myocilin was not detected in this cell line. The editing percentage at the transgenic locus ranged from 86.7% (2 ng / μl) to 97.8% (10 ng / μl) (Figure 11, Panel D), and a reduction in myocilin expression of 83% (2 ng / μl) to 87% (10 ng / μl) occurred compared to unedited controls (Figure 11, Panels B - C). These results suggest that a high editing percentage of the MYOC gene leads to a high myocilin protein knockdown.

[0179] Figure 12 demonstrates MYOC gene editing and myosin protein knockdown after delivery of LNP CTX-C12-200-CT / Cas9 mRNA / SpMCh10 sgRNA to human primary trabecular meshwork cells. In this study, human primary trabecular meshwork cells were transfected with various amounts of LNP CTX-C12-200-CT formulated with Streptococcus pyogenes Cas9 mRNA and SpMCh10 targeting the MYOC gene. Cells were transfected for 2 days, and then dexamethasone was added to the medium to induce myosin expression. Three days later, samples were collected. DNA was extracted, and PCR and TIDE were performed to determine the percentage of indels at the cleavage site at the genomic locus (Figure 12, panel A). Proteins were extracted, and immunoblotting was performed using anti-GAPDH and anti-myosin to detect wild-type myosin (Figure 12, panels B and C). Editing percentages at the genomic locus ranging from 47% (2 ng / μl) to 98% (10 ng / μl) (Figure 12, panel A) resulted in a 49% (2 ng / μl) to 85% (10 ng / μl) reduction in myosin expression compared to unedited controls (Figure 12, panels B and C). These results suggest a correlation between the editing percentage at the MYOC locus and myosin protein knockdown.

[0180] Example 5: Ex vivo LNP delivery of Cas9 and sgRNA This example describes the ex vivo delivery of Cas9 mRNA and MYOC sgRNA. Anterior segment organ cultures (ASOCs) were established from donor human cadavers with glaucoma or no reported eye conditions. The ASOCs were perfused with serum-containing medium at a flow rate of 2.5 μl / ml, and the intraocular pressure was monitored. 50 μg of LNP CTX-C12-200-CT / eGFP or LNP CTX-C12-200-CT / Cas9 mRNA / MYOC sgRNA was transfected for 24 hours (GFP) or 4 days (Cas9 / sgRNA) by introducing the formulation using a syringe and pump system. For GFP, the ASOCs were fixed, embedded, and processed for immunohistochemical (IHC) staining for GFP expression. For Cas9 mRNA / sgRNA injection, tissues (cornea, sclera, and trabecular meshwork) were isolated, and DNA or protein was isolated for TIDE or western blot analysis).

[0181] In FIG. 13, GFP protein expression in trabecular meshwork tissue after delivery of LNP CTX-C12-200-CT / eGFP mRNA in ex vivo anterior segment organ cultures (ASOCs) is demonstrated by IHC staining. The results suggest high and specific expression of GFP in trabecular meshwork tissue after LNP transfection.

[0182] Demonstration of MYOC gene editing and myosin protein knockdown in the trabecular meshwork tissue after delivery of LNP CTX-C12-200-CT / Cas9 mRNA / MYOC sgRNA in ex vivo anterior segment organ cultures (ASOCs). ASOCs were established from whole eyes provided by human cadaveric donors. 50 μg of LNP CTX-C12-200-CT formulated with Streptococcus pyogenes Cas9 mRNA and MYOC sgRNA (either SpMCh10 or SpMCh21) was injected into a perfusion system at a flow rate of 2.5 μl / ml. Four days later, the cornea, sclera, and trabecular meshwork tissue were isolated from the ASOCs. DNA was extracted and PCR and TIDE were performed to determine the percentage of indels at the cleavage site at the genomic locus (Figure 14, panel A). Proteins from experiments including donor #1656 were extracted and immunoblots were performed using anti-GAPDH and anti-myosin to detect wild-type myosin (Figure 14, panels B and C). The editing percentages in the trabecular meshwork were 27.6%, 29.7%, and 35.7% for donors 1580, 1578, and 1656, respectively; where the editing percentages in the sclera and cornea were low (1 - 9%) (Figure 14, panel A). Myosin protein expression monitored in donor 1656 showed a 44% protein reduction compared to the contralateral non-edited tissue counterpart (Figure 12, panel B and Figure 11, panel C). These results are a demonstration of the concept that MYOC gene editing in the targeted trabecular meshwork tissue results in downregulation of myosin protein in that tissue.

[0183] Example 6: In vivo LNP delivery of Cas9 and sgRNA This example describes in vivo LNP delivery of Cas9 mRNA and sgRNA in mouse eyes. ACTA2 / α-SMA was selected as a non-limiting exemplary target gene since this protein is highly expressed in the trabecular meshwork and can be readily monitored by IHC.

[0184] One microliter containing CTX-C12-CT LNP formulated with Cas9 / sgRNA was injected into the vitreous of the eyes of BALB / c mice. An sgRNA targeting the mouse ACTA2 gene (α-smooth muscle actin or SMA) was selected for high editing efficiency in vitro. Whole eyes from naive mice and mice injected with LNP were collected 1, 2, and 4 weeks after injection. Whole eyes were fixed, embedded, and processed for immunohistochemical staining for α-SMA expression. The presence of SMA in the trabecular meshwork was evaluated at 6 locations in the eye. A score from 0 (no TM cells with SMA signal) to 3 (most TM cells with SMA signal) was assigned to each of the 6 locations, and the mean score was determined for each eye.

[0185] After a single injection of LNP containing sgRNA targeting the ACTA2 mouse gene, the expression of the ACTA2 gene protein, α-SMA, decreased continuously from week 1 to week 4 as shown in Figure 15, panel A). IHC was scored for the number of cells expressing α-SMA. Figure 15, panel A shows an example of a score of 3 (the highest score) in naive / untreated animals, and Figure 15, panel B shows an example of a score of 0 (the lowest score) in the trabecular meshwork of treated animals 4 weeks after LNP injection.

[0186] This example demonstrates that LNP can deliver Cas9 mRNA / sgRNA to the trabecular meshwork in vivo, resulting in efficient editing by the Cas9 / sgRNA complex at the target sequence and downregulation of protein expression.

[0187] Sequence Listing The following table provides details for the various nucleotide and amino acid sequences disclosed herein.

[0188] [Table 5]

[0189] [Table 6-1]

Table 6-2

[0190]

Table 7

[0191]

Table 8

[0192]

Table 9

[0193]

Table 10-1

Table 10-2

Table 10-3

[0194]

Table 11

[0195]

Table 12

[0196]

Table 13-1

Table 13-2

Table 13-3

[0197]

Table 14-1

Table 14-2

Table 14-3

[0198]

Table 15

[0199] Other embodiments All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

[0200] From the above description, those skilled in the art can easily identify the essential features of the present invention and make various changes and modifications to the present invention so as to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0201] Equivalents Several progressive embodiments are described and illustrated herein. Those skilled in the art can readily envision various other means and / or structures for implementing the functions described herein and / or for obtaining the results and / or one or more advantages. Each such variation and / or modification is considered to be within the scope of the progressive embodiments described herein. More generally, those skilled in the art will appreciate that all parameters, sizes, materials, and arrangements described herein are intended to be exemplary, and that actual parameters, sizes, materials, and / or arrangements may depend on one or more specific applications in which the progressive teachings are used. Those skilled in the art can understand or confirm numerous equivalents of the specific progressive embodiments described herein using only routine experimentation. Accordingly, it is understood that the foregoing embodiments are presented by way of example only, and that progressive embodiments within the scope of the appended claims and their equivalents are specifically described and may be practiced other than as specifically claimed. The progressive embodiments of the present disclosure are directed to each of the individual characteristics, systems, products, materials, kits, and / or methods described herein. Additionally, any combination of two or more such characteristics, systems, products, materials, kits, and / or methods is included within the scope of the progress of the present disclosure if such characteristics, systems, products, materials, kits, and / or methods do not conflict with each other.

[0202] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in incorporated documents by reference, and / or the ordinary meaning of defined terms.

[0203] All references, patents, and patent applications disclosed herein are incorporated by reference for the subject matter to which each is cited, and in some cases may include the entire document.

[0204] The indefinite articles "a" and "an", as used in the specification and in the claims of this specification, should be understood to mean "at least one" unless clearly indicated otherwise.

[0205] As used in the specification and in the claims of this specification, the phrase "and / or" should be understood to mean "either or both" of the combined elements, i.e., elements that in some cases coexist and in other cases exist separately. Multiple elements listed using "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements are combined. Other elements may exist, whether or not they are related to the specifically identified elements, in addition to the elements specifically identified by the "and / or" clause. Thus, referring to, by way of non-limiting example, "A and / or B", when used in conjunction with an open-ended term such as "comprising", in one embodiment, only A (which may include elements other than B); in another embodiment, only B (which may include elements other than A); in yet another embodiment, both A and B (which may include other elements); and so on.

[0206] As used in the specification and claims herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., construed to include at least one of a number or list of elements and also more than one, and optionally may include additional unenumerated items. Terms such as "only one of" or "exactly one of" indicate otherwise clearly, or "consisting of" when used in the claims, refer to including exactly one element of a number or list of elements. Generally, as used herein, the term "or" is construed to indicate an exclusive alternative (i.e., "either one or the other but not both") only when preceded by exclusive terms such as "any one of", "one of", "only one of" or "exactly one of". When used in the claims, "consisting essentially of" has its ordinary meaning as used in the field of patent law.

[0207] As used herein, the term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation for each practice in the art. Alternatively, "about" can mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, even more preferably up to ±1% of a given value. When specific values are recited in the application and claims, the term "about" is implicit and means within an acceptable error range of the specific value in this context, unless otherwise stated.

[0208] As used in the specification and in the claims of this specification, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition is intended to allow that an element may exist whether or not specifically identified in relation to these specifically identified elements, outside of the elements specifically identified within the list of elements to which the phrase "at least one" refers. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently "at least one of A or B" or equivalently "at least one of A and / or B") may, in one embodiment, refer to at least one A, optionally including more than one, where B does not exist (and may include elements other than B); in another embodiment, may refer to at least one B, optionally including more than one, where A does not exist (and may include elements other than A); in yet another embodiment, may refer to at least one A, optionally including more than one, and at least one B, optionally including more than one (and may include other elements); and so on.

[0209] It should also be understood that, unless otherwise clearly indicated, in any method claimed in this specification that includes more than one step or operation, the order of the method steps or operations is not necessarily limited to the order in which the method steps or operations are recited.

Claims

1. A composition for delivering a CRISPR / Cas-mediated gene editing system to target trabecular meshwork cells, (a) Guide RNA for a target gene or nucleic acid encoding guide RNA; and / or (b) RNA-induced endonuclease or nucleic acid encoding an RNA-induced endonuclease A composition comprising multiple lipid nanoparticles (LNPs) complexed with a substance, thereby reducing the expression of a target gene in target trabecular meshwork cells.

2. The composition according to claim 1, wherein the target gene is the myocilin (MYOC) gene.

3. The composition according to claim 1, wherein after administration, the expression of the target gene, the expression of the protein encoded by the target gene, or both is reduced by at least 20%, at least 40%, at least 70%, or at least 90% in the eye of the subject, and / or the expression of the target gene is reduced in the trabecular meshwork cells of the eye of the subject.

4. A composition for treating glaucoma, (a) guide RNA for the myocilin (MYOC) gene or nucleic acid encoding guide RNA; and (b) RNA-induced endonuclease or nucleic acid encoding an RNA-induced endonuclease A composition comprising multiple lipid nanoparticles (LNPs) complexed with a substance, thereby reducing the expression of the MYOC gene in the target eye.

5. The composition according to claim 4, wherein the glaucoma is myocilin-associated glaucoma and / or primary open-angle glaucoma (POAG).

6. The composition according to claim 5, wherein the expression of the MYOC gene is reduced in the trabecular meshwork cells of the target eye.

7. The composition according to any one of claims 1 to 6, wherein the RNA-induced nuclease is a Cas9 nuclease, and optionally the Cas9 nuclease is a Staphylococcus aureus Cas9 (SaCas9) nuclease or a Streptococcus pyogenes Cas9 (SpCas9) nuclease.

8. The composition according to claim 7, wherein the site targeted by the guide RNA is located within exon 1, exon 2, or exon 3 of the MYOC gene.

9. The site targeted by the guide RNA is: (i) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 27 and 55 to 115; (ii) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109 and 113-115; and / or (iii) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76 and 115; and, Guide RNA as needed: (i) A spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-27 and 55-115; (ii) A spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 6, 10, 15, 18, 26, 59, 61, 63, 64, 66, 69, 72-77, 79, 81, 82, 90, 95, 98-101, 104, 106, 107, 109 and 113-115; (iii) A spacer sequence having an RNA sequence corresponding to any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 10, 64, 73, 74, 75, 76, and 115; (iv) SaCas9 sgRNA or SpCas9 sgRNA; (v) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195-371; and / or (vi) A nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371, The composition according to claim 8.

10. One of the LNPs comprises an ionizable cationic lipid, a helper lipid, a sterol, and a poly(ethylene glycol) lipid (PEG lipid), wherein the LNP optionally comprises about 20-60% ionizable lipid, about 18.5-60% sterol, about 0.01-30% helper lipid and about 0-10% PEG lipid. Furthermore, as appropriate, (i) The ionizable cationic lipid is selected from the group consisting of C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA, and DLin-KC2_DMA; (ii) Helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and diundecanoylphosphatidylcholine (DU PC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimirystoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE, 18:0-18:1 Selected from the group consisting of PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoylphosphatidylglycerol (DOPG), and dipalmitoyl-sn-glycero-3-PG (DPPG); (iii) The sterol is selected from the group consisting of cholesterol, sitosterol, β-sitosterol, phytosterol, fucosterol, animal sterols and ergosterol, or the sterol is selected from the group consisting of cholesterol, sitosterol, β-sitosterol, campesterol, stigmasterol, fucosterol and ergosterol; and / or (iv) The PEG lipid is DMG-PEG, DSG-PEG, PEG ceramide, or PEG phospholipid. The composition according to any one of claims 1 to 6.

11. The composition according to any one of claims 1 to 6, wherein one of the plurality of LNPs comprises about 50 mol% C12-200, DLIN-MC3, DODMA or DOTAP, about 10 mol% DSPC, about 37.0 to 39.5 mol% cholesterol or sitosterol and about 0.5 to 3.0% DMG-PEG; optionally, the LNP comprises about 50 mol% C12-200, about 10 mol% DSPC, about 37.0 to 39.5 mol% sitosterol and about 0.5 to 1.5% DMG-PEG; and optionally, the average particle size of the plurality of LNPs is about 80 to 100 nm or 85 to 95 nm.

12. (i) A plurality of LNPs to be administered to a subject by intravitreous injection or anterior chamber injection; (ii) The composition is for a single dose of multiple LNPs to a subject; (iii) MYOC expression in the target eye is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after use; (iv) Myosirin protein in trabecular meshwork cells of the eye in question is reduced by at least 20%, at least 40%, at least 70%, or at least 90% after use; and / or (v) The subject is a human being, The composition according to any one of claims 1 to 6.

13. The composition according to any one of claims 1 to 6, wherein the LNPs are separately complexed with (a) guide RNA or a nucleic acid encoding guide RNA, and (b) RNA-induced endonuclease or a nucleic acid encoding RNA-induced endonuclease, and optionally, the LNP complexed with (a) guide RNA or a nucleic acid encoding guide RNA and the LNP complexed with (b) RNA-induced endonuclease or a nucleic acid encoding RNA-induced endonuclease are different LNPs.

14. A system for treating a subject having glaucoma, (i) gene editing methods that target the reduction of myocilin (MYOC) gene expression in the target eye; and (ii) Lipid nanoparticles (LNPs) that deliver gene editing methods to the target eye. A system that includes this.

15. The system according to claim 14, wherein the glaucoma is myocilin-associated glaucoma and / or primary open-angle glaucoma (POAG).

16. The system according to claim 15, wherein the gene editing means is CRISPR / Cas-mediated gene editing, and optionally the CRISPR / Cas-mediated gene editing comprises an RNA-induced nuclease and a guide RNA that targets a site in the MYOC gene, and optionally the RNA-induced nuclease is a Cas9 nuclease, and optionally the Cas9 nuclease is a Staphylococcus aureus Cas9 (SaCas9) nuclease or a Streptococcus pyogenes Cas9 (SpCas9) nuclease.

17. The system according to claim 16, which is to be administered to a subject by intravitreous injection or anterior chamber injection.

18. The site targeted by the guide RNA is (i) located within exon 1, exon 2, and exon 3 of the MYOC gene; (ii) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-27 and 55-115; and / or (iii) A nucleotide sequence selected from the group consisting of SEQ ID NOs: 64, 73, 74, 75, 76 and 115, Guide RNA as appropriate (i) It is SaCas9 sgRNA or SpCas9 sgRNA; (ii) comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 195-371; and / or (iii) A nucleotide sequence selected from the group consisting of SEQ ID NOs: 258, 267-270, 309, 319, 328-331, 370, and 371, The system according to claim 14.

19. The LNP comprises ionizable cationic lipids, helper lipids, sterols and poly(ethylene glycol) lipids (PEG lipids), wherein the LNP optionally comprises about 20-60% ionizable cationic lipids, about 18.5-60% sterols, about 0.01-30% helper lipids, and / or about 0-10% PEG lipids. Furthermore, as appropriate, (i) The ionizable cationic lipid is selected from the group consisting of C12-200, cKK-E12, DLIN-MC3, DLIN-MC4, DLIN-MC5, DODMA, DOTAP, DODAP, DC cholesterol, DLin-DMA, DLin-K-DMA, and DLin-KC2_DMA; (ii) Helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and diundecanoylphosphatidylcholine (DU PC), phosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimirystoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE, 18:0-18:1 Selected from the group consisting of PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), dioleoylphosphatidylglycerol (DOPG), and dipalmitoyl-sn-glycero-3-PG (DPPG); (iii) The sterol is selected from the group consisting of cholesterol, sitosterol, phytosterol, fucosterol, animal sterol and ergosterol, or the sterol is selected from the group consisting of cholesterol, sitosterol, β-sitosterol, campesterol, stigmasterol, fucosterol and ergosterol; and / or (iv) The PEG lipid is DMG-PEG, DSG-PEG, PEG ceramide, or PEG phospholipid. The system according to any one of claims 14 to 18.

20. The system according to any one of claims 14 to 18, wherein the LNP comprises about 50 mol% C12-200, DLIN-MC3, DODMA or DOTAP, about 10 mol% DSPC, about 37.0 to 39.5 mol% cholesterol or sitosterol and about 0.5 to 3.0% DMG-PEG, and optionally the LNP comprises about 50 mol% C12-200, about 10 mol% DSPC, about 37.0 to 39.5 mol% sitosterol and about 0.5 to 1.5% DMG-PEG.